Technical Abstract: Puccinia striiformis causes stripe rust on wheat, barley, and many grass species. Co-dominant microsatellite markers are more suitable for studying population structures of this dikaryotic fungus. In this study, we characterized simple sequence repeat (SSR) loci based on three previously developed expressed sequence tag (EST) libraries for P. striiformis f. sp. tritici (Pst), the wheat stripe rust pathogen. SSR loci were isolated by screening the EST sequences using the Simple Sequence Repeat Identification Tool. By scanning 3,311 unique EST sequences, filtering out those with repeat sequences less than 18 bp, and screening those with suitable flanking sequences, 46 were selected for designing primers using the Primer3 program. The 46 primer pairs were tested on races PST-1, PST-21, and PST-127 of Pst and race PSH-45 of P. striiformis f. sp. hordei using the M13 tailing and fluorescent capillary electrophoresis on an ABI3730 genotyper. The three PST races represent the most diverse races of the wheat stripe rust pathogen from 1960s to 2007 in the U.S. Thirty-four primer pairs produced repeatable polymorphic bands and 19 of them generated co-dominant markers among the four races. The lengths of the amplicons ranged from 130 to 506 bp containing 5 to 13 di-, tri-, or tetra-nucleotide repeats. The SSR primers should be useful in studying the population structure and evolution of the pathogen.